Three-dimensional visualization of the lymphatic, vascular and neural network in rat lung by confocal microscopy

In order to demonstrate the intricate interconnection of pulmonary lymphatic vessels, blood vessels, and nerve fibers, the rat lung was selected as the target and sliced at the thickness of 100 μm for multiply immunofluorescence staining with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), alpha smooth muscle actin (α-SMA), phalloidin, cluster of differentiation 31 (CD31), and protein gene product 9.5 (PGP9.5) antibodies. Taking the advantages of the thicker tissue section and confocal microscopy, the labeled pulmonary lymphatic vessels, blood vessels, and nerve fibers were demonstrated in rather longer distance, which was more convenient to reconstruct a three-dimensional (3D) view for analyzing their spatial correlation in detail. It was clear that LYVE-1⁺ lymphatic vessels were widely distributed in pulmonary lobules and closely to the lobar bronchus. Through 3D reconstruction, it was also demonstrated that LYVE-1⁺ lymphatic vessels ran parallel to or around the α-SMA⁺ venules, phalloidin⁺ arterioles and CD31⁺ capillaries, with PGP9.5⁺ nerve fibers traversing alongside or wrapping around them, forming a lymphatic, vascular and neural network in the lung. By this study, we provide a detailed histological view to highlight the spatial correlation of pulmonary lymphatic, vascular and neural network, which may help us for insight into the functional role of this network under the physiological and pathological conditions.